Stem cells have the ability to differentiate into two or more cell types while possessing the capacity of self-renewal. Studies are currently underway to harvest stem cells from adipose tissue as well as bone marrow. Adipose-derived stem cells isolated from adipose tissue are multipotent stem cells that have the ability to differentiate into various cell types, including adipocytes, myocytes, chondrocytes and osteocytes.
Adipose stem cells have the advantages of superior self-renewal capacity and easy culture ex vivo when compared to marrow-derived mesenchymal stem cells. Other advantages of adipose stem cells are that they can be isolated from abundant adipose tissue and can be harvested in a simple and safe manner. From a functional standpoint, adipose stem cells and marrow-derived mesenchymal stem cells were found to share almost the same characteristics, including multipotency, cytological characteristics, immunological characteristics and tissue regeneration. That is, adipose stem cells have been considered as possible replacements for mesenchymal stem cells and have proven their great usefulness in medical applications.
In comparison with mesenchymal stem cells, adipose stem cells are advantageous in easy and safe tissue acquisition and unlimited tissue supply and demand. In addition, adipose stem cells are easy to culture ex vivo. Due to these advantages, adipose stem cells may be considered in preference to marrow-derived mesenchymal stem cells in terms of tissue accessibility, stability, effectiveness and economic efficiency.
Many methods have been proposed to isolate and culture adipocytes. According to a typical method, after fat is sucked out or excised and crushed to pieces, its tissue is digested with collagenase, followed by centrifugation. Then, the sediment is collected and a stromal vascular fraction (SVF) is separated therefrom. The SVF is used for cell culture.
Further, according to the method disclosed in Korean Patent No. 788632, an adipose-containing suspension in physiological saline obtained from human adipose tissue by liposuction is homogeneously re-suspended in an appropriate amount of physiological saline, a proper amount of the resulting suspension is placed in a flask or roller bottle for cell culture, followed by stationary culture or roller bottle culture. In the case of stationary culture, the suspension is allowed to stand for at least 6 to 12 hours and then the cell layers attached on the surface of the flask are treated with trypsin and collected. At this time, the suspended matter is directly collected in a small amount of physiological saline and is used without further processing. Alternatively, when it is intended to reduce the volume of the cell layers, the cell layers collected in the physiological saline are subjected to centrifugation at 1000 rpm for 10 minutes and the settled pellet layers only are used. The separated cell layers contain adult stem cells and fibroblasts. The separated cell layers are mixed with fat to prepare a composition for skin care or plastic surgery. The composition contains adult stem cells, fibroblasts and fat or adipocytes.
On the other hand, PCT International Publication No. WO2005/042730 suggests a method for preparing stem cells without collagenase treatment, the method comprising: A) obtaining an aspirate by liposuction; B) transferring the aspirate to a centrifuge to obtain a cell fraction; C) subjecting the cell fraction to centrifugation by specific gravity; and D) collecting a cell layer with lower specific gravity than that of erythrocytes.
A container for enzymatic treatment and centrifugation that is currently used in clinical applications is in the form of a tube whose one end is completely closed and includes a stopper fastened to the outer surface of the tube on top thereof. When it is desired to move a content of the tube or to feed a washing solution into a container containing adipose-derived stem cells, a substance separated from the content is removed out of the tube or the washing solution enters the container in a state in which the tube is in an open position.
As such, adipose-derived stem cells as active ingredients are obtained by sucking out or excising fat, discharging it with a washing solution, followed by centrifugation. In addition, the active ingredients must be mixed with other ingredients for use in secondary applications.
Some serious problems may be caused in this course. The first problem is that it is not easy to actually isolate active ingredients by centrifugation. It is clinically known that 500 thousand cells can be isolated from about 100 cc of adipose tissue aspirate. This value corresponds to only about one millionth of the total number of cells. Accordingly, methods using centrifugation are inefficient. That is, it takes a long time for isolation by centrifugation and extreme conditions for isolation do great damage to effective cells.
Another problem is that in the course of the treatment of fat extracts, it is not easy to mix with other ingredients.
Another problem may be fatal. That is, during acquisition and isolation of fat and extraction, culture and reinfusion of active ingredients, cells may be exposed to the outside, posing the risk of microbial contamination. This problem adversely affects the safety of humans, such as side effects resulting from secondary infection or contamination.
Specifically, as soon as the stopper connected to the container is removed, active ingredients in the container come into contact with large quantity of air and may be contaminated by falling dirt and dust. In attempts to solve these problems, stem cells are clinically isolated in clean rooms of Class 1000 or above. However, much cost and time are required to ensure safety. In most cases, clinicians remove the isolated substances while identifying the layers with their eyes to separate the desired layer. In this course, loss of the active ingredients is inevitable.